Antibiotic, balhimycin, a process for its production and its use as pharmaceutical

ABSTRACT

Balhimycin, a compound of the molecular formula C66H73C12N9024, is a novel Glycopeptide antibiotic and has the following structure:   &lt;IMAGE&gt;   &lt;IMAGE&gt;

This is a division of application Ser. No. 07/996,936, filed Dec. 30,1992, now U.S. Pat. No. 5,451,570 which is a continuation-in-part ofapplication Ser. No. 07/735,891, filed Jul. 25, 1991, now abandoned.

This invention relates to a new glycopeptide antibiotic named Balhimycinfrom an Actinomycete culture number Hoechst India Limited Y-86,21022.Balhimycin may be described as an antibacterial antibiotic belonging tothe glycopeptide class. Glycopeptide antibiotics are narrow-spectrumantibiotics acting mainly against Gram positive bacteria. Their activityagainst methicillin resistant S. aureus (MRSA) strains make them avaluable drug in treating infections caused by MRSA. They are alsouseful as growth promoters in the veterinary field. Glycopeptideantibiotics are described in Topics in Antibiotic Chemistry, Vol. 5,page 119 (1980), Journal of Antibiotics, Vol. 38, page 561 (1985),Journal of Antibiotics Vol. 40, page 924 (1987), Journal of OrganicChemistry, Vol. 54, page 983 (1989).

However, Balhimycin described herein differs from all the knownglycopeptide antibiotics in its molecular formula and therefore formsthe subject of this invention. Furthermore a Chemical Abstracts on-linesearch performed with the search keys of molecular weight and molecularformula confirms the novelty of the compound.

The microorganism, culture No. Hoechst India Limited, Y-86,21022,henceforward referred to as Y-86,21022, used for the production ofBalhimycin was isolated from a soil sample collected at Thamu forest,Himalaya, India. The microorganism, Y-86,21022, belongs to the orderActinomycetales. The microorganism Y-86,21022 has been deposited withDeutsche Sammlung von Mikroorganismen under the conditions of the Treatyof Budapest on Apr. 6, 1990 and it has received No. DSM 5908.

A further aspect of the present invention is to provide a process forthe production of the new antibiotic Balhimycin from culture No. HoechstIndia Limited Y-86,21022, its mutants and variants. The said processcomprises cultivation of culture Y-86,21022, its mutants and variantsunder aerobic conditions in a nutrient medium containing sources ofcarbon and nitrogen, nutrient inorganic salts and trace elements andisolation and purification of the said antibiotic from the culturebroth. The carbon sources may be starch, glucose, sucrose, dextrin,fructose, molasses, glycerol, lactose or galactose. The preferred carbonsource is glycerol. The sources of nitrogen may be soyabean meal, peanutmeal, yeast extract, beef extract, peptone, malt extract, corn steepliquor, gelatin or casamino acids. The preferred nitrogen source issoyabean meal. Nutrient inorganic salts may be sodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, calciumchloride, calcium carbonate, potassium nitrate, ammonium sulphate ormagnesium sulphate. Trace elements could be salts of iron, manganese,copper, zinc or cobalt or other heavy metals.

Cultivation of culture No. Y-86,21022 is preferably carried out attemperatures between 28° and 32° C. and ph between 6.0 and 8.0. Inparticular culture No. Y-86,21022 is cultivated at 29° C. (±1° C.) andph about 7.0.

The fermentation is carried out preferably for 60 to 72 hours whenoptimal yield of the antibiotic of the present invention is obtained.Fermentation is particularly carried out for 68-72 hours under submergedconditions in shake flasks as well as in laboratory fermenters. Ifdesired, an anti-foam agent such as Desmophen® (Polyols, Bayer-AGLeverkusen, West Germany) can be used in the fermenters. The progress offermentation and formation of the compound according to presentinvention can be detected by High Pressure Liquid Chromatography (HPLC)and by measuring the bioactivity of the culture broth againstStaphylococci species by the known microbial agar plate diffusion assaymethod. The preferred culture is Staphylococcus aureus 3066 known to beresistant to Methicillin, a beta-lactam antibiotic reported in theliterature.

Balhimycin can for example be isolated from the culture broth by directadsorption on suitable adsorbants like activated carbon, Diaion HP-20®(high porosity resin based on a polystyrene-divinylbenzenecopolymer--Mitshubishi Chemical Industries, Japan) or Amberlite®XAD(porous resin based on polystyrene-acrylic acid ester--Rohm & Haas Co.,U.S.A.). The preferred adsorbent is Diaion HP-20. Balhimycin can beeluted out of these adsorbents using mobile phases such as water,methanol, acetone, acetonitrile or suitable combinations thereof. Thepreferred eluants are aqueous methanol or acetone.

The aforementioned active eluates containing Balhimycin can beconcentrated and can be further purified in a number of ways. Forexample, readsorption and elution processes with activated charcoal,Amberlite XAD-4 and 7, Diaion HP-20; gel filtration with SephadexLH-20®, or G series gels (Pharmacia Fine Chemicals AB, Sweden) usingwater, methanol, acetone or appropriate combinations thereof as eluants;ion-exchange chromatography with IRC-50 (H⁺) at Ph range of 6.5-8.5using 0.1N Hcl as the eluant; or medium pressure liquid chromatography(MPLC) on suitable adsorbents like silica, modified silica such asreverse phase silica, for example octadecyldimethylsilylated silica(RP-18), neutral alumina. Furthermore countercurrent chromatography witha given multicomponent solvent system may also be used for the saidpurpose. The preferred method of purification includes repeated MPLC onRP-18 using aqueous methanol or acetonitrile containing suitableadditive such as salt or acid, as the eluant.

Balhimycin can be converted into its pharmacologically acceptable salts,for example, with inorganic and organic acids such as HCl, H₂ SO₄,citric acid, lactic acid, succinic acid, acetic acid, trifluoro aceticacid in a known manner.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the structure of Balhimycin.

FIG. 2 shows the HPLC retention time of Balhimycin.

FIG. 3 shows the ¹ H NMR of Balhimycin at 400 MHz D₂ O, 300K.

FIG. 4 shows the ¹³ C-NMR of Balhimycin in dry DMSO, 320K.

FIG. 5 shows the ¹³ C-NMR spectrum of Balhimnycin.

The physico-chemical and spectral properties of Balhimycin are shown inTable 1 below. The structure of Balhimycin is shown in FIG. 1.

                  TABLE 1                                                         ______________________________________                                        Physics-chemical and spectral properties of Balhimycin                        (as its trifluoroacetate salt)                                                ______________________________________                                        Nature            White powder                                                Chemical type     Basic glycopeptide of the                                                     vancomycin class                                            Solubility        Water, dimethylsulfoxide                                    M. pt.            >300° C. (decomposition)                             [α].sub.D.sup.22                                                                          -23.2 ± 2° (C = 5, H.sub.2 O)                     High Pressure Liquid                                                                            4.2 minutes, 4 × (250 + 30)                           Chromatography (HPLC)                                                                           mm 10μ "ODS-Hypersil"                                    Retention Time    column, eluant 18%                                                            acetonitrile in water containing                                              0.1% TFA, flow rate 2 ml/                                                     minute, detection 220 nm, chart                                               speed 10 mm/minute, FIG. 2 of                                                 the accompanying drawings                                   Molecular Formula C.sub.66 H.sub.73 Cl.sub.2 N.sub.9 O.sub.24                                   determined by high resolution                                                 FAB mass spectrometry                                                         (M + H.sup.+  measured m/z                                                    1446.4204, calculated m/z                                                     1446.4205 for                                                                 .sup.12 C.sub.66.sup.1 H.sub.74.sup.35 Cl.sub.2.sup.14                        N.sub.9.sup.16 O.sub.24)                                    UV.sub.max (H.sub.2 O)                                                                          208, 285 nm                                                 .sup.1 H NMR (400 MHz D.sub.2 O, 300K)                                                          FIG. 3 of the accompanying                                                    drawings                                                    .sup.13 C-NMR (Dry DMSO, 320K)                                                                  FIG. 4 of the accompanying                                                    drawings                                                    .sup.13 C-NMR-Spectrum                                                                          FIG. 5 of the accompanying                                                    drawings                                                    ______________________________________                                    

Balhimycin and its physiologically tolerated salts can be administered,for example, orally, intramuscularly or intravenously. Pharmaceuticalswhich contain Balhimycin as active substance are subject of the presentinvention also. They can be prepared by mixing the said compound withone or more pharmacologically tolerated auxiliaries and/or excipientssuch as, for example, fillers, emulsifiers, lubricants, maskingflavours, colorants or buffer substances, and converted into a suitablepharmaceutical form such as, for example, tablets, coated tablets,capsules, or a suspension or solution suitable for parenteraladministration.

Examples of auxiliaries and/or excipients which may be mentioned aretragacanth, lactose, talc, agar-agar, polyglycols, ethanol and water.Suitable and preferred for parenteral administration are suspension orsolutions in water. It is also possible to administer the activesubstances as such, without vehicles or diluents, in a suitable form,for example in capsules.

Suitable doses of the compound of this invention or its physiologicallytolerated acid addition salts are about 0.1 to 20 g/day, preferably 0.5to 4 g/day, for an adult of body weight about 60 kg.

It is possible to administer single doses or, in general, multipledoses, it being possible for the single dose to contain the activesubstance in an amount of about 50 to 4,000 mg, preferably of about 500to 2,000 mg.

The instant invention is further characterized by the following examplesand by the content of the patent claims.

EXAMPLE 1 Isolation of the culture Y-86,21022 from soil

(a) Composition of nutrient isolation medium

    ______________________________________                                        Corn starch           10.0   g                                                Casein                1.0    g                                                Peptone               1.0    g                                                Yeast Extract         1.0    g                                                K.sub.2 HPO.sub.4     0.5    g                                                Agar powder           13.0   g                                                Demineralized water   1.0    liter                                            Ph                    7.5                                                     ______________________________________                                    

(b) Soil plating and isolation

10 g of soil collected from Thamu Forest, Himalaya, India were added to90 ml of sterilized demineralized water in a 250 ml Erlenmeyer flaskwhich was shaken for 2 hours on a rotary shaker (220 rpm). The abovesoil suspension was then serially diluted in steps of 10 upto (10⁻⁵).From the last dilution, 1 ml of suspension was placed at the centre of asterile glass petri plate (15 cms diameter) to which was then pouredapproximately 50 ml of the above isolation medium supplemented with 25mcg/ml of amphotericin B as anti fungal agent and cooled to 45° C. andthe plate swirled thoroughly. The mixture of soil suspension and mediumwas allowed to settle and incubated at 28° C. (±1° C.) for 7 days. Thepetri plate was periodically observed and the culture No. Y-86,21022 wasisolated from amongst the growing microorganisms.

EXAMPLE 2 Maintenance of the culture Y-86,21022 Composition ofmaintenance medium

Culture No. Y-86,21022 was maintained on the following medium

    ______________________________________                                        Malt Extract          10.0   g                                                Yeast Extract         4.0    g                                                Glucose               4.0    g                                                Agar Powder           13.0   g                                                Demineralized water   1.0    liter                                            pH                    7.0                                                     ______________________________________                                    

After dissolving the ingredients thoroughly by heating, it wasdistributed in test tubes and then sterilized at 121° C. for 20 minutes.The test tubes were cooled and allowed to solidify in a slantingposition. The agar slants were streaked with the growth of the cultureNo. Y-86,21022 by a wire loop and incubated at 28° C. (±1° C.) until agood growth was observed. The well grown cultures were stored in therefrigerator at +8° C.

EXAMPLE 3 Fermentation of culture Y-86,21022 in shake flasks Compositionof seed medium I

    ______________________________________                                        Glucose                15.0   g                                               Soyabean meal          15.0   g                                               Corn steep liquor      5.0    g                                               CaCO.sub.3             2.0    g                                               Demineralized water    1000   ml                                              pH                     7.0                                                    ______________________________________                                    

The above seed medium was distributed in 80 ml amounts in 500 mlErlenmeyer flasks and autoclaved for 20 minutes, The flasks were cooledto room temperature and each flask was then inoculated with a loopful ofthe above mentioned well grown culture of Example 2 and shaken on arotary shaker for 72 hours at 240 rpm at 29° C. (±1° C.) to give seedculture.

Composition of the production medium

    ______________________________________                                        Glycerol               15.0   g                                               Soyabean meal          10.0   g                                               CaCO.sub.3             1.0    g                                               NaCl                   5.0    g                                               CoCl.sub.2             0.001  g                                               Demineralized water    1000   ml                                              pH                     7.0                                                    ______________________________________                                    

The production medium was distributed in 60 ml amounts in 500 mlErlenmeyer flasks and autoclaved at 121° C. for 20 minutes. The flaskswere cooled and then inoculated with the above mentioned seed culture(1% v/v). The fermentation was carried out on a rotary shaker at 240 rpmand at a temperature of 29° C. (±1° C.) for 68 hours.

The production of the antibiotic was determined by HPLC and by testingthe bioactivity against S. aureus 3066 using the well diffusion methodin a known manner. After harvesting, the culture broth was centrifugedand Balhimycin isolated from the culture filtrate and purified asdescribed in Example 4.

EXAMPLE 4 Cultivation of the culture No. Y-86,21022 in fermenters

Preparation of seed culture in shake flasks

The seed medium of Example 3 was distributed in 160 ml amounts in 1 LErlenmeyer flasks and autoclaved for 20 minutes. The seed culture wasgrown in these flasks as described in Example 3.

Large Scale Fermentation

    ______________________________________                                        Glycerol               15.0   g                                               Soyabean meal          10.0   g                                               CaCO.sub.3             1.0    g                                               NaCl                   5.0    g                                               Demineralized water    1000   ml                                              pH                     6.8                                                    ______________________________________                                    

100 liters of the production medium in 150 liter Marubishi fermenteralong with 30 ml of Desmophen as antifoam was sterilized in situ for 24minutes at 121° C. cooled to 29°±1° C. and seeded with 3 liters of theseed culture mentioned above.

The fermentation was run with the following parameters:

    ______________________________________                                        Temperature        29° C. (±.5° C.)                          Agitation          110-120 rpm                                                Aeration           80-100 epm                                                 Harvest time       69-72 hrs                                                  ______________________________________                                    

The production of the antibiotic was monitored by the bioactivityagainst S. aureus 3066. When fermentation was discontinued, the pH ofthe culture broth was 6.0-7.0. The culture broth was centrifuged afterharvesting and the antibiotic Balhimycin was isolated and purified fromthe culture filtrate as described below.

Isolation and purification of Balhimycin

Approximately 100 L of the harvested broth, as obtained in Example 4,was separated from the mycelium by centrifugation. The resulting brothfiltrate (80 L, pH 6.5) was passed through a column of 5 L of 1 DiaionHP-20, in water. The column was washed with 40 L of demineralized waterafter which the washings were colourless. The column was then washedwith 8 L of 1M aqueous NaCl followed by 10 L of demineralized water.This process was repeated once. The column was then washed with 9 L of30% MeOH in water and finally eluted with 51 L of 75% MeOH in watercollected in fractions of 1 L size. The presence of Balhimycin in theseeluates was monitored by its activity against Staphylococcus aureus 3066which is a methicillin resistant organism. The combined active eluateswere concentrated under vacuo at 40°-45° C. to approximately 1 L whichwas then lyophilized to give 38 g of crude Balhimycin as a powder.

The above mentioned crude material was then subjected in three batchesof 10 g each to medium pressure liquid chromatography (MPLC) on RP-18packed in a 6.5×55 cm glass column having a bed volume of 1.2 L. Thecolumn was eluted with 2 L of water containing 0.1% trifluoroacetic acid(TFA), followed by elution with 5% acetonitrile (4 L), 7.5% acetonitrile(9 L) and 10% acetonitrile (24.5 L) in water containing 0.1% TFA.Balhimycin eluted out in 10% acetonitrile eluates which were collectedin fractions of 500 ml and monitored by in vitro antibiotic activity.The combined active eluates were concentrated under high vacuum at 40°C. and then lyophilized to give 2.1 g of Balhimycin as a pale yellowpowder. Three such processes gave a total of 6.3 g of semi-pureBalhimycin from the 30 g of the crude material.

The 6.3 g of semi-pure Balhimycin thus obtained was finally purified bysubjecting in three batches of 2.1 g each to MPLC on RP-18 in a 6.0×45mm glass column with a bed volume of 500 mL. The column was washed with1 L of water containing 0.1% TFA followed by 2 L and 1.5 L of 5% and7.5% acetonitrile respectively in water containing 0.1% TFA. Balhimycineluted out in 10% acetonitrile in water containing 0.1% TFA, thefractions of 250 ml each were monitored both by a UV detector working at220 nm and by in-vitro antibiotic assay. A total of 8 L of activeeluates were concentrated under high vacuum at 40° C. followed bylyophilization to give 600 mg of Balhimycin as a trifluoroacetate saltin the form of white powder. From the 6.3 g of semi-pure Balhimycin, 1.8g of pure Balhimycin was procured.

Biological properties of Balhimycin

The antibacterial activity of Balhimycin as MIC values required toinhibit the growth of various bacteria are shown in Table II.

                  TABLE II                                                        ______________________________________                                        Minimum inhibitory concentrations (MIC)                                       of Balhimycin trifluoroacetate                                                Test Organism        MIC (mcg/ml)                                             ______________________________________                                        S. aureus 209 P      0.39                                                     S. aureus (MethR)    0.39                                                     S. aureus 3066 (MethR)                                                                             0.78                                                     S. aureus E 690 (MethR)                                                                            0.39                                                     Streptococcus faecalis ATCC 29212                                                                  1.56                                                     S. faec. D 21777     0.78                                                     S. faec. D Endococcen                                                                              0.78                                                     S. faecium D-59      1.56                                                     S. faecium D-65      0.78                                                     Staph. epidermidis 178                                                                             1.56                                                     S. epi. 825          3.12                                                     S. epi. 823 (Teicoplanin.sup.R)                                                                    12.5                                                     Staph. haemolyticus 712                                                                            1.56                                                     S. haemo. 809 (Teicoplanin.sup.R)                                                                  6.25                                                     E. coli 9632         >100                                                     P. vulaaris          >100                                                     Ps. aeruaingu        >100                                                     ______________________________________                                    

We claim:
 1. A process for the preparation of Balhimycin ##STR2## whichcomprises fermentation of Actinomycete species Y-86,21022 (DSM 5908),and the mutants thereof, cultivated under aerobic conditions in anutrient medium containing sources of carbon and of nitrogen, inorganicnutrient salts and trace elements, andisolation and purification of thecompound from the culture broth.
 2. The process as claimed in claim 1,wherein the cultivation is carried out at temperatures between about 28°and 32° C. and at a pH between about 6 and
 8. 3. The process as claimedin claim 1, wherein the cultivation is carried out at 29° C. (±1° C.)and a pH about 7.0.
 4. The process as claimed in claim 1, wherein thefermentation is carried out for 68 to 72 hours.
 5. The process asclaimed in claim 1, wherein the fermentation is carried out at submergedfermentation.
 6. The process of claim 1 wherein said carbon source isselected from the group consisting of starch, glucose, sucrose, dextrin,fructose, molasses, glycerol, lactose or galactose.
 7. The process ofclaim 6 wherein said carbon source is glycerol.
 8. The process of claim1 wherein said nitrogen source is soyabean meal, peanut meal, yeastextract, beef extract, peptone, malt extract, corn steep liquor, gelatinor casamino acids.
 9. The process of claim 8 wherein said nitrogensource is soyabean meal.
 10. Isolated Actinomycete species Y-86,21022(DSM 5908).